免疫荧光层析快速检测河豚毒素的研究

本研究利用自制的河豚毒素荧光免疫层析检测卡对河豚毒素进行快速定量检测,并分析其稳定性、灵敏度、趋势线拟合程度及检测范围。研究结果表明,自制的河豚毒素荧光免疫层析检测卡变异系数为0.124,拟合方程相关系数r~2值为0.995 5,检测卡的检测范围为2~600 ng/m L,可以满足对河豚毒素快速定量检测的要求。     

河鲀毒素诱导菊黄东方鲀膜转运蛋白基因的表达

为探究参与河鲀肝脏和皮肤累积河鲀毒素(Tetrodotoxin, TTX)的过程及相关调控基因,按每克体质量河鲀0.25μg TTX剂量尾部肌肉注射人工繁育的菊黄东方鲀(Takifugu flavidus),发现肝脏中TTX含量在注射后4 h时最高(19.12 ng/g),24 h下降到8.92 ng/g, TTX主要在肝实质细胞中累积;皮肤中TTX含量一直处于上升趋势,4 h为13.70 ng/g, 24 h为22.91 ng/g; TTX主要在皮肤表皮层、基底层细胞中累积。比较对照组和注射24 h后肝脏和皮肤转录组发现：TTX注射导致肝脏组织338个基因上调、350个基因下调;而皮肤组织有598个基因上调,同时有595个基因下调;RT-qPCR验证slc2a12、slc5a7、slc25a22、slc25a15、slc35a3、arap1、ugt2a2、ggt5、psbp2等膜转运蛋白相关基因在注射TTX后在肝脏和皮肤中的表达水平显著上调,可能与TTX累积过程有关。     

河豚毒素的分离纯化研究

本实验根据河豚毒素的理化性质,分别采用去除河豚内脏油脂、除蛋白、活性炭吸附、阳离子交换、分子筛层析、乙醇沉淀等方法得到纯度较高的河豚毒素。本方法生产河豚毒素具有对人体无毒无害、操作工艺简单、生产成本低廉、易于工业化批量生产等特点,并且生产出的河豚毒素可以基本满足各种对河豚毒素应用的需求,是较为可行的生产河豚毒素的方法。     

利用固定化草酸氧化酶快速检测河豚毒素

本研究旨为快速简便地检测河豚鱼体中河豚毒素(Tetrodotoxin,TTX)提供可操作手段。将河豚毒素用氢氧化钠处理,得到定量的草酸盐。利用戊二醛法将草酸氧化酶固定于聚丙烯膜上,通过比色法检测草酸含量,可以测算出河豚毒素的含量。结果显示,检测限量为0.80μg.g-1,线性范围为1.44~14.40μg.g-1,加标平均回收率为88.54%,变异系数在11%以内。实验证实,该方法可抗河豚体内多种组分的干扰。利用该法测定3组暗纹东方(Takifugu obscurus)肝脏中河豚毒素含量,并将检测结果与小鼠生物试验进行对照,相关性良好。该方法检测时间短,对仪器设备要求低,便于推广应用。     

Tetrodotoxin as a tool to elucidate sensory transduction mechanisms: the case for the arterial chemoreceptors of the carotid body

Carotid bodies (CBs) are secondary sensory receptors in which the sensing elements, chemoreceptor cells, are activated by decreases in arterial PO(2) (hypoxic hypoxia). Upon activation, chemoreceptor cells (also known as Type I and glomus cells) increase their rate of release of neurotransmitters that drive the sensory activity in the carotid sinus nerve (CSN) which ends in the brain stem where reflex responses are coordinated. When challenged with hypoxic hypoxia, the physiopathologically most relevant stimulus to the CBs, they are activated and initiate ventilatory and cardiocirculatory reflexes. Reflex increase in minute volume ventilation promotes CO(2) removal from alveoli and a decrease in alveolar PCO(2) ensues. Reduced alveolar PCO(2) makes possible alveolar and arterial PO(2) to increase minimizing the intensity of hypoxia. The ventilatory effect, in conjunction the cardiocirculatory components of the CB chemoreflex, tend to maintain an adequate supply of oxygen to the tissues. The CB has been the focus of attention since the discovery of its nature as a sensory organ by de Castro (1928) and the discovery of its function as the origin of ventilatory reflexes by Heymans' group (1930). A great deal of effort has been focused on the study of the mechanisms involved in O(2) detection. This review is devoted to this topic, mechanisms of oxygen sensing. Starting from a summary of the main theories evolving through the years, we will emphasize the nature and significance of the findings obtained with veratridine and tetrodotoxin (TTX) in the genesis of current models of O(2)-sensing.     

Tetrodotoxin sensitivity of the vertebrate cardiac Na+ current

Evolutionary origin and physiological significance of the tetrodotoxin (TTX) resistance of the vertebrate cardiac Na(+) current (I(Na)) is still unresolved. To this end, TTX sensitivity of the cardiac I(Na) was examined in cardiac myocytes of a cyclostome (lamprey), three teleost fishes (crucian carp, burbot and rainbow trout), a clawed frog, a snake (viper) and a bird (quail). In lamprey, teleost fishes, frog and bird the cardiac I(Na) was highly TTX-sensitive with EC(50)-values between 1.4 and 6.6 nmol·L(-1). In the snake heart, about 80% of the I(Na) was TTX-resistant with EC(50) value of 0.65 μmol·L(-1), the rest being TTX-sensitive (EC(50) = 0.5 nmol·L(-1)). Although TTX-resistance of the cardiac I(Na) appears to be limited to mammals and reptiles, the presence of TTX-resistant isoform of Na(+) channel in the lamprey heart suggest an early evolutionary origin of the TTX-resistance, perhaps in the common ancestor of all vertebrates.     

Tetrodotoxin-bupivacaine-epinephrine combinations for prolonged local anesthesia

Currently available local anesthetics have analgesic durations in humans generally less than 12 hours. Prolonged-duration local anesthetics will be useful for postoperative analgesia. Previous studies showed that in rats, combinations of tetrodotoxin (TTX) with bupivacaine had supra-additive effects on sciatic block durations. In those studies, epinephrine combined with TTX prolonged blocks more than 10-fold, while reducing systemic toxicity. TTX, formulated as Tectin, is in phase III clinical trials as an injectable systemic analgesic for chronic cancer pain. Here, we examine dose-duration relationships and sciatic nerve histology following local nerve blocks with combinations of Tectin with bupivacaine 0.25% (2.5 mg/mL) solutions, with or without epinephrine 5 μg/mL (1:200,000) in rats. Percutaneous sciatic blockade was performed in Sprague-Dawley rats, and intensity and duration of sensory blockade was tested blindly with different Tectin-bupivacaine-epinephrine combinations. Between-group comparisons were analyzed using ANOVA and post-hoc Sidak tests. Nerves were examined blindly for signs of injury. Blocks containing bupivacaine 0.25% with Tectin 10 μM and epinephrine 5 μg/mL were prolonged by roughly 3-fold compared to blocks with bupivacaine 0.25% plain (P < 0.001) or bupivacaine 0.25% with epinephrine 5 μg/mL (P < 0.001). Nerve histology was benign for all groups. Combinations of Tectin in bupivacaine 0.25% with epinephrine 5 μg/mL appear promising for prolonged duration of local anesthesia.     

Geographic Variations in the Toxin Profile of the Xanthid Crab Zosimus aeneus in a Single Reef on Ishigaki Island,Okinawa, Japan

Toxic crabs of the family Xanthidae contain saxitoxins (STXs) and/or tetrodotoxin (TTX), but the toxin ratio differs depending on their habitat. In the present study, to clarify within reef variations in the toxin profile of xanthid crabs, we collected specimens of the toxic xanthid crab Zosimus aeneus and their sampling location within a single reef (Yoshihara reef) on Ishigaki Island, Okinawa Prefecture, Japan, in 2018 and 2019. The STXs/TTX content within the appendages and viscera or stomach contents of each specimen was determined by instrumental analyses. Our findings revealed the existence of three zones in Yoshihara reef; one in which many individuals accumulate extremely high concentrations of STXs (northwestern part of the reef; NW zone), another in which individuals generally have small amounts of TTX but little STXs (central part of the reef; CTR zone), and a third in which individuals generally exhibit intermediate characteristics (southeastern part of the reef; SE zone). Furthermore, light microscopic observations of the stomach contents of crab specimens collected from the NW and CTR zones revealed that ascidian spicules of the genus Lissoclinum were dominant in the NW zone, whereas those of the genus Trididemnum were dominant in the CTR zone. Although the toxicity of these ascidians is unknown, Lissoclinum ascidians are considered good candidate source organisms of STXs harbored by toxic xanthid crabs.     

河豚毒素及其代谢产物的研究进展

对河豚毒素及其代谢产物的理化性质、分析方法、在动物体内的分布及产生来源等进行了简单的概述,旨在为研究含毒动物体内积累河豚毒素的机制提供一些理论根据。